Table 1.

Strains, plasmids, and primers used in this study

Strain, plasmid, or primerRelevant properties or sequenceaReference or origin
E. coli
 BL21(DE3) E. coli B; F ompTrB mB (λDE3) 37
 SA1503(DE3) lon-100 his-87 ompT::Kmr relA1 rpsL781 spoT1 thi-1 (λDE3)19a
P. aeruginosa
 PAO1PrototrophB. H. Holloway
 PAO198PAO1 withfabF::Gmr This study
 PAO204PAO1 with fabD(Ts)This study
Plasmids
 pEX100TApr sacB +; gene replacement vector 34
 pUCGMAprGmr; source of Gmrcassette 33
 pWSK29 and pWKS30Apr; low-copy-number cloning and T7 expression vectors 44
 pCYB1Apr; intein-chitin binding domain expression vectorNew England Biolabs
 pET-15bApr; hexahistidine fusion and expression vectorNovagen
 pGEM-TApr; TA-cloning vectorPromega
 pNamApr; low-copy-number intein-chitin binding domain expression vector19a
 pT7-7Apr; T7 promoter expression vector 39
 pPS671Apr fabF+ pabC + (ligation of chromosomal 4-kb EcoRI-HindIII fragment between the same sites of pWSK29)This study
 pPS681Apr fabD+ fabG+ acpP+fabF+ pabC + (ligation of 3.4-kbBamHI-EcoRV fragment from pPS840 between theBamHI and XbaI [blunt-ended] sites of pPS671)This study
 pPS831Apr; PCR-amplified 147-bp genomic segment from PAO1 cloned into pGEM-TThis study
 pPS840Apr fabD+fabG+ acpP + (3.7-kb chromosomalEcoRV-BamHI fragment cloned between the same sites of pWSK29)This study
 pPS966Apr; ligation of 1.8-kb NdeI-PstI fragment from pPS981 between the same sites of pT7-7This study
 pPS981Apr acpP +; ligation of NdeI-SapI-digested PCR fragment between the same sites of pCYB1This study
 pPS1096Apr acpP + (ligation of 735-bpHindIII-XbaI fragment from pPS966 between the same sites of pNam)This study
Primers
 ACP1ATCGAGGAGCG(A/C/T/G)GT(G/C)AAGAAGAT(A/C)AT
 ACP2GTCGAACTCCTC(G/C)TC(A/C/T/G)AG(G/C)GCCAT
 FabD1TGGATGCTCTCCACCtgGCGACCCGGGCTGT
 ACP-Nde NdeI-TGAAAACAACATatgAGCACCATCGAAGAACGCGTT
 ACP-Sap SapI-ATCCGGCTCTTCCGCAttgCTGGGGAGC
 FabD-Nde NdeI-GGGACCTATcatATGTCTGCATCCCTCGCATTCGTC
 FabD-Bam BamHI-CCCAGGatCCCCAGTTCCAGCGCAATCGCC
  • a Primer sequences are printed 5′ to 3′; lowercase letters indicate nonmatching oligonucleotides used to either form the indicated motif as underlined or introduce other nonmotif changes indicated in lowercase letters.